B6 pituitary tumor cells via a plasma membrane version of estrogen receptor-α

Steroids 64 (1999) 5–13

Rapid actions of estrogens

Rapid actions of estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of estrogen receptor-a Cheryl S. Watsona,*, Andrea M. Norfleeta, Todd C. Pappasa, Bahiru Gametchub a

Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX, USA b Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA Manuscript received September 19, 1998; accepted November 12, 1998

Abstract The focus of our work on rapid actions of estrogens has been on the immuno-identification of a membrane version of the estrogen receptor-a (mERa) and the correlation of the presence of this receptor to the rapid secretion of prolactin in pituitary tumor cells. We demonstrated the mERa by both fluorescence and immuno-enzyme-cytochemistry and with both conventional and confocal microscopy in the cell line GH3/B6 and its sublines. Its presence on cells (including recently subcloned ones) is very heterogenous, unlike the nuclear ERa, which is present in every cell. An impeded ligand (estradiol covalently linked to BSA) binds to mERa and elicits the same response. A total of eight antibodies to ERa recognize mERa, making it likely that the membrane and nuclear proteins are highly related. Immunoidentification techniques have also been used to identify mERa on the MCF-7 human breast cancer cell line. Estradiol at very low concentrations elicits prolactin release from GH3/B6 cells within a few minutes of application. This response is bimodal, with effective concentrations in both the picomolar and nanomolar ranges. Prolactin release is also elicited or inhibited by ERa-specific antibodies. The characteristics of mERa and the membrane receptor for glucocorticoids have many similarities, suggesting that this mode of subcellular location/function alternative might be used by other members of the gene family. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Estrogen; Membrane steroid receptor; Estrogen receptor-a; Prolactin release; Pituitary; Breast cancer

1. Introduction The story of steroid receptors serving as ligand-activated transcription factors is well described for all members of this protein family and is very well accepted. However, when the field was just beginning to gather information on the nature of the proteins that mediate effects of steroids and similar ligands, it was also proposed that steroids might, in addition, act through ligand-activated membrane receptors [1–3]. Various technical problems, along with the trends of science, led to this aspect of the mechanism of action of steroids not being actively pursued. However, a number of laboratories have recently been compelled to reopen investigations on how steroids could cause very rapid effects that do not require RNA and protein synthesis [4 – 8].

Our own investigations into the membrane estrogen receptor of pituitary tumor cells were encouraged by two considerations: the successful immuno-identification of membrane glucocorticoid receptors on lymphoma cells by one of us [9] and by a study clearly demonstrating the rapid electrophysiological effects of estrogen in GH3/B6 cells [10]. Since this latter study found that the changes in firing rate and membrane resistance occurred within a minute or two of estrogen application to these cells, we thought that such cells would be an appropriate model in which to ask if membrane-located estrogen receptors existed, and if they were part of the machinery necessary for rapid responses to the hormone.

2. Experimental * Corresponding author. Dr. Cheryl S. Watson, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-0645, USA. Todd C. Pappas is currently at the Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USA.

2.1. Cell growth GH3/B6 cells were a gift of Dr. Bernard Dufy (Universitie de Bordeaux II, Bordeaux, France). Cells were rou-

0039-128X/99/$–see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 9 - 1 2 8 X ( 9 8 ) 0 0 1 - 7 - X

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C.S. Watson et al./Steroids 64 (1999) 5–13

tinely cultured in serum-supplemented media composed of Ham’s F-10 (Gibco-BRL, Gaithersburg, MD, USA), 12.5% heat-inactivated horse serum (Gibco-BRL; Hyclone, Logan, UT, USA) and 2.5% heat-inactivated defined/supplemented bovine calf serum (Hyclone). Defined medium (DM1) was adapted from Hayashi and Sato [11], and consisted of phenol red-free RPMI 1640 (Gibco-BRL), insulin (10 mg/mL, Boehringer Mannheim, Indianapolis, IN, USA), bovine transferrin (5 mg/mL, Boehringer Mannheim), parathyroid hormone (bovine 1–34, 0.5 ng/mL, Bachem, Torrance, CA, USA), thyrotropin-releasing hormone (TRH, 1 ng/mL, Bachem), 3,39,5-triiodo-L-thyronine (3 3 10211 M, Sigma, St. Louis, MO, USA), and basic fibroblast growth factor (1 ng/mL, Boehringer Mannheim). Later experiments (enzyme-immunochemistry, prolactin [PRL] release assays) employed a much simplified defined medium (DIB) containing DMEM (GIBCO-BRL), insulin-transferrin-selenium (Sigma), and 0.1% BSA (Sigma). Immunoseparation of MCF-7 cells was achieved by immunopanning as described previously [9,12]. 2.2. Live-cell labeling with fluorescent-tagged secondary antibody Characterization and affinity purification of the polyclonal anti-peptide antibodies (Abs) to ER (R3 and R4), has been described previously [13]. Monoclonal Abs H222 and H226 and polyclonal Ab ER21 were a gift of Dr. Geoffrey Greene [14 –18]. Abs H151 (anti-human hinge region) and C542 (anti-human carboxy terminus) are from StressGen Inc. For immunocytochemical studies using polyclonal Abs (both generated in rabbit), cells were cultured in DM1 for 2 d on poly-D-lysine (180,000 mol wt, Sigma) treated coverslips (12 mm in diameter, Baxter, McGaw Park, IL, USA) placed in 24-well tissue culture plates (Corning Glass Works, Corning, NY, USA). R3 (1:100 dilution) or ER21 (10 mg/mL) and Cy3-conjugated goat anti-rabbit Ab (1:100, Jackson ImmunoResearch, West Grove, PA, USA) were pre-incubated in phosphate-buffered saline (PBS) 1 1% BSA (PBSA) with 4% normal goat serum (Jackson ImmunoResearch) for 30 min on ice. Live cells on glass coverslips were incubated in ice-cold PBS for 15 min. The coverslips were inverted onto 15 ml of the Ab mixture for 30 min, then washed four times with PBSA, fixed with 4% paraformaldehyde for 5 min, mounted on glass slides in 20% glycerol/80% PBS, and sealed with clear nail polish. Fluorescence micrographs were taken on a Leitz Orthomat Microscope using a 633 oil immersion objective lens. We have previously shown that labeling cells live and at 4° prohibits access of the Ab complex to the intracellular compartment [13]. For immunocytochemistry using monoclonal Abs generated in rat [13], an amplifying Ab was used. The chilled cells were incubated with 10 mg/mL of the primary Ab diluted in 1% PBSA. Cells were washed and fixed in 4% paraformaldehyde, then blocked for 1 h in 1% PBSA. A 1:5000 dilution of rabbit anti-rat secondary Ab

(Cappel, Organon Teknika, Durham, NC, USA) was placed on the cells for 20 min. The cells were then washed three times, blocked with 4% normal goat serum and incubated in a 1:100 dilution of Cy3-conjugated goat anti-rabbit Ab (Jackson ImmunoResearch). To label nuclear estrogen receptor, cells were gently fixed for 1 min with 1% paraformaldehyde prior to labeling, and were permeabilized with 0.1% (vol/vol) Triton X-100 (Sigma) in PBS for 10 min. Reactive groups were quenched with 3% PBSA for 1 h. Coverslips were inverted for 30 min onto primary Ab diluted in 1% PBSA, and then washed four times in 1% PBSA. For ER21, the cells were treated with 4% normal goat serum for 20 min, then inverted onto the secondary Abs for 20 min. For monoclonal Abs, cells were incubated for 20 min with a rabbit anti-rat Ab (diluted 1:5000), washed three times in 1% PBSA, incubated for 20 min in 4% normal goat serum, and then in Cy3-conjugated goat anti-rabbit for 20 min. 2.3. Confocal scanning laser microscopy These studies employed a Noran Odyssey Confocal microscope (Noran Instruments, Middleton, WI, USA). Image capture, processing, and stepper motor functions were all executed with Image-1/AT software (version 4.0, Universal Imaging, West Chester, PA, USA). The captured images were reconstructed on a Silicon Graphics R4000-50 VGX workstation using VoxelView Ultra software (version 2.0.1, Vital Images, Fairfield, IA, USA). The VoxelView program was used to interpolate a single section between each optical section to reconcile resolution in the z dimension to that in the x and y dimensions, and to emphasize features of the staining. Contour lines were traced around the perimeter of the stained area every third section as an aid to orientation and viewing cell surface features. 2.4. Labeling with E2-BSA-FITC 1,3,5 [10] Estratriene-3,17b-diol-6-one 6 carboxymethoxime: bovine serum albumin (E2-BSA) was obtained from Steraloids (Wilton, MA, USA; molar ratio E2:BSA 5 35:1). E2-BSA-FITC (molar ratio E2:BSA 5 10:1) was obtained from Sigma. 17b-estradiol (E2, Sigma) was diluted in ethanol and then further diluted in phenol red-free RPMI 1640 to a final E2 concentration of 100 nM–1 mM (final ethanol concentration of 0.01%). GH3/B6 cells were cultured as for immunocytochemistry. Cells were chilled 15 min in phenol red-free RPMI 1640 with 200 mg/mL BSA, then washed three times in chilled RPMI 1640. Coverslips were inverted onto 15 ml of E2-BSA-FITC in RPMI 1640 and incubated at 4° for 30 min. After the labeling, cells were fixed for 1 min (to minimize autofluoresence) in 4% paraformaldehyde and mounted as described above. Photomicrographs using T-MAX 400 film (Eastman Kodak Co., Rochester, NY, USA)

C.S. Watson et al./Steroids 64 (1999) 5–13

2.5. Fixed cell staining with enzyme-immunocytochemistry GH3/B6/F10 pituitary tumor cells [13] or MCF-7 breast cancer cells were cultured on glass coverslips that had been treated with poly-D-lysine (Sigma) for 72 h in the defined medium DIB. The cells were washed once in phosphatebuffered saline, pH 7.4 (PBS), prior to fixation. In order to render the cell membranes impermeable to Ab, a 30-min fixation period in 1% glutaraldehyde at room temperature was employed. After fixation, the cells were washed three times in PBS. Subsequent incubations were carried out at room temperature, using reagents from a VECTASTAIN ABC-Alkaline Phosphatase kit in conjunction with the Vector Red substrate (Vector Labs, Burlingame, CA, USA). The fixed cells were blocked for 20 min in PBS that contained 1.5% normal horse serum (NHS). Cells were then incubated for 60 min in the presence or absence of primary Ab diluted in 1.5% NHS. After a PBS wash, cells were incubated for 30-min in the presence of a biotinylated “universal” horse anti-mouse/anti-rabbit IgG diluted in PBS, then washed with PBS and exposed to an avidin-biotinylated alkaline phosphatase complex for 30 min. The cells were rinsed twice with PBS over a 10-min period, and then incubated with VectorRed substrate and levamisole (Vector Labs), an inhibitor of endogenous alkaline phosphatase, for 2 min. The reaction was stopped with an excess volume of water, and methyl green was used to counterstain the cells during a 2-min incubation at 55°. The cells were dehydrated through a series of washes in ethanol and xylene, prior to mounting with Cytoseal 280 (Stephens Scientific, Riverdale, NJ, USA). Photomicrographs were obtained using an Olympus AHBS microscope, equipped with a fluorescence attachment (Model AH2-RFL) and camera. Brightfield photomicrographs were taken with Kodak Royal Gold film. 2.6. PRL release assays—radioimmunoassay for PRL GH3/B6/F10 cells, cultured in 24-well plates for 72 h in DIB, were washed once and pre-incubated for 15 min in DMEM containing 20 mM HEPES and 0.1% BSA (DHB). The cells were maintained at 37°C for the pre- and the test incubations. The pre-incubation medium was removed and replaced at time zero with test medium (DHB 1/2 0.001% EtOH 1/2 test agents). The test agents included 172 b estradiol (E2) and the anti-ER2 Abs. A stock solution of E2 (Sigma) in 95% EtOH was diluted such that the final EtOH concentration was 0.001%, which was also used in all control and test conditions. After 3 or 6 min, test medium was collected into prechilled tubes, which were immediately stored at 220°. PRL concentrations in the test media were determined by radioimmunoassay using reagents provided by the National Institute of Diabetes and Digestive and Kidney Disease and the National Hormone and Pituitary Program. The intra-assay variability was 7%. PRL release observed in the vehicle control group was considered to be 100%. A one-way analysis of variance (ANOVA) compar-

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ing treatment groups to the control was performed using SigmaStat version 2.0 (Jandel, San Rafael, CA, USA); post hoc group differences were analyzed using Dunnett’s Test. Statistical significance was accepted at p , 0.05.

3. Results and discussion 3.1. Immuno-identification as a tool for characterizing mER There are inherent difficulties in trying to demonstrate the binding of a labeled lipophilic ligand to a protein residing in a lipophilic structure such as the plasma membrane. Partitioning of the steroid into the lipid bilayer often obscures the binding of the labeled ligand to proteins in that membrane. Thus, use of specific Abs in identifying membrane steroid receptors presented itself as a useful alternative technique once Abs to these low abundance regulatory proteins were available. As the cDNAs for the receptors were cloned and sequenced, the difficulty of purifying adequate quantities of protein for raising Abs to them disappeared, and anti-peptide Abs made these reagents more plentiful. So we began by creating an anti-peptide, hinge region-specific Ab to the rat intracellular receptor we now know as ERa. Our first experiments used a peptide affinity column-purification of our polyclonal Ab and a secondary anti-rabbit Ab linked to cy3 fluorescent probe [12,13,19,20]. We used live cell immunolabeling at 4° to prevent Ab from entering the cells. We observed punctate, asymmetric labeling on these cells (Figure 1), which was capable of patching, capping, and disappearing from cells when the incubation temperature was elevated to 37° for 15 min to 1 h. We also observed that there was great heterogeneity among the cells of this clonal GH3/B6 cell line, that cells were labeled to quite varying degrees, and some cells were not labeled at all (Figure 1). When cells were permiabilized with detergents (or occasionally accidently by manipulations during immunocytochemistry) all cells in the culture displayed nuclear estrogen receptor (Figure 2). This suggested that either cells rapidly mutated their ability to express this protein, or that the mER was dynamically regulated. To answer the question of whether our Ab had accidently recognized a protein completely unrelated to the intracellular estrogen receptor simply because we chose an epitope by chance shared with another protein, we then tried a series of other Abs to the rat and human estrogen receptor, as they became available. At present count, 8 Abs representing 6 epitopes have recognized the estrogen receptor on GH3/B6 cells and their various sublines (see below). Figure 3 summarizes the Abs we have used, to date, to visualize mER. We have also recently explored use of other immunocytochemical techniques to identify and visualize mER on cells. In order to have a different view, and perhaps new insight into the characteristics of mER, to reduce our sole reliance on fluorescence microscopy, and to amplify the

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C.S. Watson et al./Steroids 64 (1999) 5–13

Fig. 1. Labeling of estrogen receptor-a on live GH3/B6 cells. Cells were labeled at 4° with affinity-purified R4 antibody (Ab) and anti-rabbit-Cy3 secondary Ab. Fluorescence micrograph (A) and phase contrast micrograph (B) of the same field. 633.

Fig. 2. All cells have intracellular estrogen receptor-a. Photomicrographs of cells that were fixed and made permeable with detergent prior to R4 antibody labeling. Fluorescence (A) and phase contrast (B) photomicrographs of the same field. 633.

Ab-identified signal for greater sensitivity, we developed protocols using light microscopy and Ab-conjugated enzyme-producing colored reaction products to investigate mER. These fixation techniques do not allow Abs inside cells enough to see the more plentiful nuclear ER, which sometimes interferes with visualizing the membrane form. Again, there is heterogeneity of mER expression on cells, and the nature of the staining is punctate (Figure 4). Since this technique allows us to fix cells before the application of Ab, we can now assume that the punctate labeling (which suggests clustering of receptors in discreet areas) is not just an artifact of the multivalency of Abs binding to receptors and aggregating them. We have also observed this type of labeling of mER on MCF-7 breast cancer cells (Figure 5). We also used the tool of confocal microscopy to further address the question of subcellular localization. Modern

confocal analysis systems allow resolution in individual optical planes (sections of the cell), and so provide better distinctions between plasma membrane and nuclear labeling. Spatial separation of signals allowed by this technique reduces the flare created by nearby signals; this has been a problem when the signal of bright nuclear staining spreads to other areas of the cell and sometimes obscures the weaker, membrane signals. Figure 6a shows optical sectioning of a cell stained with fluorescently labeled Ab, and the accompanying Figure 6b shows the 3-dimensional reconstruction of these signals into a whole cell image. The contour lines of the sections are drawn to show the edges (plasma membrane) of the cell. Again, the mER signal is very patchy and unevenly distributed over the surface of the cell, and the technique employed further confirms a plasma

C.S. Watson et al./Steroids 64 (1999) 5–13

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Fig. 3. Epitopes recognized by anti-estrogen receptor (ER)-a antibodies (Abs) that also recognize membrane ERa. Relationship to the established functional domain structure of intracellular ERa is shown. Ab sources and descriptions are described in the Experimental section.

membrane location distinct from the nuclear location seen in permiabilized cells. Finally, we wondered if the Ab recognition of the ER antigen on cell surfaces was synonymous with the protein which bound and responded to estradiol. Therefore, we did double-labeling experiments with an impeded ligand, estradiol covalently attached to BSA (E2-BSA), to determine if it could bind to mER simultaneously with ER Ab. The heterogeneous pattern of binding on cells allowed us to colocalize the two labels visualized with different color fluors (Figure 7). Furthermore, E2-BSA was capable of eliciting the PRL release response (see below) rapidly, and at low concentrations [12]. 3.2. The rapid response to estrogens Rapid effects of estrogen at the membrane are interesting in and of themselves, but are more interesting if connected to some cellular function. Secretion of peptide hormones (such as PRL) are a prominent feature of this cell type, and these peptide hormones, in turn, have important functional consequences. The activity of secretagogues are traditionally very rapid. Certainly estrogen stimulates PRL synthesis in pituitary cells at a relatively slow rate [21,22], but there was also the possibility that regulation of secretion of PRL was by a separate, more rapid mechanism. Therefore, we tested estrogen for PRL secretagogue activity over a time frame synonymous with the previously observed membrane perturbations [10]. We were able to observe the release of a significant bolus of PRL as soon as 1-min after the application of estrogen by radioimmunoassay (RIA) [13]. To see if the presence of the identified estrogen receptor was associated with this function, we separated the heterogeneous

wild-type GH3/B6 cells into mER1 vs. mER2 populations by two techniques: immunopanning [19] and limiting dilution subcloning (13). In both cases the mER-enriched cell populations (mER1) demonstrated a robust, rapid release of PRL in response to estradiol, while the mER2 cell populations did not. Thus, mER seemed necessary for the estrogen-induced secretory response. It is frequently observed that some membrane-initiated responses have been demonstrated with only pharmacological levels of hormone. Therefore, we tested the range of sensitivity of the secretory response to estrogens, to determine if such functions could be mediated by physiological amounts of hormones. Early experiments indeed showed that subnanomolar estradiol could mediate the release response [13]. However, upon further optimization of the system with the mER1 subclone F10, and use of a very simple defined medium (adding only insulin, transferrin, selenium and BSA to DMEM) we discovered a further sensitivity of the response in the subpicomolar range (Figure 8). Interestingly, this response is bimodal, that is, two separate peaks of activity are noted (one in the picomolar range and the other in the nanomolar range). It appears that this is one of the most sensitive responses to estradiol ever described. Furthermore, others are now beginning to describe highly sensitive nongenomic responses to steroids [23,24]. Because there was a report in the literature that an antiidiotypic Ab to estrogen was capable of eliciting a rapid response to estrogens, in this case the cellular uptake of calcium in osteoblasts [25], we wondered if any of the Abs that recognized mER on GH3/B6 cells would likewise mediate or modulate the PRL release response. Table 2 lists Abs that we have used in PRL release assays. Hinge region-

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C.S. Watson et al./Steroids 64 (1999) 5–13

Fig. 4. Immunolabeling of glutaraldehyde fixed GH3/B6/F10 cells by anti-estrogen receptor-a antibody H151. 2003.

specific Abs R3 and R4 [13] both can elicit PRL release (at several concentrations) while the hinge region-specific Ab H151 (Stressgen) inhibits estrogen-induced PRL release. Several other Abs recognizing epitopes in the N-terminal (ER21, H226) ligand binding (H222, [18]) or C-terminal (C542, Stressgen) did not affect PRL release measured at 3 to 6 min after Ab or estradiol application (manuscript submitted). Therefore, the binding of a molecule that recognizes, and can alter, the shape of the estrogen receptor can also mimic or inhibit functions mediated by that receptor. Investigations of the nuclear version of ER and other steroid receptors have instructed us in the importance of conforma-

tional changes in the activities of these proteins [26 –30]. Not surprisingly, it would appear that the same must be the case for the membrane version of this protein.

3.3. Generalities about membrane steroid receptors and further considerations In our experience, one of the striking things about the membrane estrogen receptor is that many of its general characteristics are similar to those of the membrane glucocorticoid receptor. Table 1 gives a brief indication of this

Fig. 5. Immunolabeling of glutaraldehyde fixed MCF-7 cells that had been selected by two consecutive rounds of immunopanning. Anti-estrogen receptor-a antibody H151. 2003.

C.S. Watson et al./Steroids 64 (1999) 5–13

Fig. 6. Confocal analysis of membrane estrogen receptor-a (ERa). Serial optical sections show that anti-ERa antibody R3 localizes with the perimeter of the cell. A well-attached cell with punctate labeling and peripheral antigen localization is shown using five sections along the z axis (1–5) and with a reconstructed image (B) rotated 30° from horizontal. Solid lines show the perimeter of the cell for every third section. From Pappas TC, Gametchu B, Watson CS. Membrane estrogen receptors identified by multiple antibody and impeded ligand labeling. FASEB J 1995;9:404 –10. Reprinted with permission.

comparison, listing the characteristics and citing the references in which each was described. Both membrane glu-

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Fig. 7. E2-BSA-FITC binding and membrane estrogen receptor immunoreactivity colocalize in live-labeled cells. (A) Fluorescence micrograph of E2-BSA-FITC-labeled cells. (B) Corresponding R3-Cy3 labeling. (C) Accompanying phase-contrast photomicrograph. 633.

cocorticoid receptor (mGR) and mER can be recognized by Abs raised to their intracellular counterparts. This suggests

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C.S. Watson et al./Steroids 64 (1999) 5–13 Table 2 Comparison of the characteristics of membrane estrogen receptor (mER) versus the membrane glucocorticoid receptor (mGR)

Fig. 8. Rapid prolactin (PRL) release elicited by a broad range of E2 concentrations. Hormonal treatments were measured against a control containing 0.001% ethanol vehicle (C). PRL was collected from the medium for 3 min after hormone application. Each point represents mean 6 SEM for 3 experiments, each consisting of 4 – 8 samples/concentration. *p ,0.05 vs. the control.

a theme in steroid receptor structure-function that we predict will be exploited by other hormonal classes of steroid receptors and their related family members. The membrane versions of these steroid receptors have both been shown to be larger than their intracellular counterparts by Western and biotin-blot technologies. Immunological identity plus a larger size suggests that the uniqueness of these receptors lies in added components, either sequence or posttranslational modifications. When visualized by immunocytochemistry, both receptors appear to be punctate and exhibit characteristics of proteins that are mobile in the membrane. Both receptors are very vulnerable to enzymatic digestion and biotinylation, and multiple Abs seems to have equal access to them; these characteristics suggest that they are completely on the outside of the cell, not buried to any large extent in the membrane. The appearance of the proteins in clustered groups on the cell surface suggests their association with each other or with other distinct membrane substructures. Sequestration of membrane signaling molecules in such substructures has been previously described [31]. Impeded ligands are capable of both labeling and eliciting Table 1 Bioactivity of antibodies to estrogen receptor-a in the rat pituitary tumor cell subline, GH3/B6/F10

Antibody

Epitope location

Species specificity

Effect on prolactin release

R4; R3 H151 C542 H226 D75

Hinge region Hinge region C-terminus DNA binding region Ligand binding region

Rat, human Rat, human Rat, human Rat, human Human

Increase Decrease None None None

Characteristic

mGR

mER

Size (mol wt.) larger Appearance punctate Patches and caps Cell shape difference 1 vs. 2 cells Multiple epitopes recognized by Abs to nuclear receptor Surface biotin labeling Trypsin sensitive (antigen and function) Impeded ligand binding and response Serum and ligand regulation Cell cycle regulation Specific RNA In normal (immature) cells In other cancer cells Ab triggers function

86–210 Yes Yes Yes 3

60–200 Yes Yes Yes 8

Yes Yes Yes? Yes Yes Yes Yes Not tested Not tested

Yes Yes Yes Yes Yes? Yes? Not tested Yes Yes

Where experiments are preliminary, a question mark is placed next to the entry. References to individual characteristics are cited in the text. Abs, antibodies.

responses from these receptors, showing that they are both compartmentally and functionally distinct from the corresponding nuclear receptors. Their distinct preference for stages of the cell cycle, stages of development, and up- or down-regulation via mechanisms that we do not yet fully understand (such as by serum components), results in the tremendous heterogeneity of their expression, even in recently subcloned cell lines. This suggests that membrane steroid receptors are a very dynamic, highly regulated population of receptors. When we finally are able to characterize what controls their expression, and thus their functions, we will undoubtedly have a very valuable new tool for therapeutic manipulation and understanding of hormonal mechanisms in general. Acknowledgments The authors gratefully acknowledge Dr. Thomas J. Collins for iodination of prolactin, Dr. David Konkel for critical reading of this manuscript, and the Department of Pharmacology, University of Texas Medical Branch for use of their microscope facilities. Financial support for CSW has been from NICHD grant 32481 and the John Sealy Memorial Endowment Fund. BG has been supported by NCI grant 65674 and by the Midwest Athletes Against Cancer (MAAC) Fund. References [1] Pietras RJ, Szego CM. Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature 1997;265:69 –72. [2] Szego CM, Davis JS. Adenosine 3’,5’-monophosphate in rat uterus: Acute elevation by estrogen. Proc Natl Acad Sci USA 1967;58: 1711– 8.

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[20] Pappas TC, Gametchu B, Watson CS. Membrane estrogen receptors identified by multiple antibody and impeded ligand labeling. FASEB J 1995;9:404 –10. [21] Amara JF, Dannies PS. 17 beta-Estradiol has a biphasic effect on GH cell growth. Endocrinology 112:1141–3. [22] Chun TY, Gregg D, Sarkar DK, Gorski J. Differential regulation by estrogens of growth and prolactin synthesis in pituitary cells suggests that only a small pool of estrogen receptors is required for growth. Proc Natl Acad Sci USA 1998;95:2325–30. [23] Santillan GE, Boland RL. Studies suggesting the participation of protein kinase A in 1,25(OH)(2)-vitamin D-3-dependent protein phosphorylation in cardiac muscle. J Mol Cell Cardiol 1998;30:225– 33. [24] Endoh H, Sasaki H, Maruyama K, Takeyama K, Waga I, Shimizu T, Kato S, Kawashima H. Rapid activation of MAP kinase by estrogen in a bone cell line. Biochem Biophys Res Commun 1997;235:99 – 102. [25] Somjen D, Kohen F, Lieberherr M. Nongenomic effects of an antiidiotypic antibody as an estrogen mimetic in female human and rat osteoblasts. J Cell Biochem 1997;65:53– 66. [26] Elliston JF, Katzenellenbogen BS. Comparative analysis of estrogen receptors covalently labeled with an estrogen and an antiestrogen in several estrogen taget cells as studied by limited proteolysis. J Steroid Biochem 1998;29:559 – 69. [27] Allan GF, Leng X, Tsai SY, Weigel NL, Edwards DP, Tsai MJ, O’Malley BW. Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation. J Biol Chem 1992;267:19513–20. [28] Beekman JM, Allan GF, Tsai SY, Tsai MJ, O’Malley BW. Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain. Mol Endocrinol1993; 7:1266 –74. [29] Leng X, Tsai SY, O’Malley BW, Tsai MJ. Ligand-dependent conformational changes in thyroid hormone and retinoic acid receptors are potentially enhanced by heterodimerization with retinoic X receptor. J Steroid Biochem Mol Biol 1993;46:643– 61. [30] Gass EK, Leonhardt SA, Nordeen SK, Edwards DP. The antagonists RU486 and ZK98299 stimulate progesterone receptor binding to deoxyribonucleic acid in vitro and in vivo, but have distinct effects on receptor conformation. Endocrinology 1988;139:1905–19. [31] Wu C, Butz S, Ying Y, Anderson RG. Tyrosine kinase receptors concentrated in caveolae-like domains from neuronal plasma membrane. J Biol Chem 1997;272:3554 –9.